The high speed supernatant (cytosol) of Novikoff hepatoma cells contains factors which are capable of increasing the rate of DNA synthesis by the Novikoff cytoplasmic, rat liver cytoplasmic or nuclear DNA polymerases several fold. These factors are relatively inactive with heterologous and rat liver mitochondrial DNA polymerases. They are present in very low levels in normal liver. Five such factors have been found, and three have been partially purified. The proposed research is aimed at 1) identifying any and all factors participating in or controlling DNA replication; 2) purifying each factor to homogeneity; 3) characterizing each factor with regard to enzymatic activity (if any), molecular weight and subunit structure, stoichiometry of its action on in vitro DNA synthesis, binding to DNA or DNA polymerase, interaction with other factors, subcellular localization and amount present under different physiological conditions; 4) clearly establishing a role for each in DNA replication in vivo. The purification scheme will follow established methods for protein purification (salt and acid precipitation, column chromatography, gel filtration, affinity chromatography, isoelectric focusing). The criterion for homogeneity will be a single band in polyacrylamide gels and localization of the activity to that band by elution from sliced, unstained sister gels. Each factor will be assayed for phosphodiesterase, phosphatase, endonuclease, exonuclease, RNA polymerase, DNA ligase, ATPase, DNA polymerase and DNA unwinding activity. Binding to DNA or DNA polymerase will be determined by gel filtration. Subcellular localization will be studied with labeled antibody techniques. Attempts will be made to isolate replication complexes by sarcosyl-Mg banding in sucrose gradients or by indirect antibody precipitation and to study the component proteins.